Diagnostic assay for anti-von willebrand factor cleaving protease (adamts13) antibodies

ABSTRACT

This invention relates to a kit to be used in an assay system for determination of an anti-von Willebrand Factor-cleaving protease (“anti-vWF-cp”) antibody in a sample. The kit comprises vWF-cp and/or vWF-fragment(s) immobilized on a solid phase. The kit can be used in a method for determination of anti-vWF-cp antibodies from a patient, for the diagnosis of disorders associated with the occurrence of anti-vWF-cp-antibodies, and the differentiation of various forms of thrombotic microangiopathy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 10/422,052, filed Apr. 22, 2003, which application is hereinincorporated by reference.

FIELD OF THE INVENTION

This invention relates to a kit to be used in an assay system fordetermination of an anti-von Willebrand Factor-cleaving protease(ADAMTS13) antibody (“anti-vWF-cpantibody”) in a sample suspected tocomprise an anti-vWF-cp antibody. The kit can be used in a method fordiagnosis of disorders associated with the occurrence ofanti-vWF-cp-antibodies in patients, and to discriminate betweendifferent forms of thrombotic microangiopathy.

BACKGROUND OF THE INVENTION

One important protein in primary hemostasis is von Willebrand Factor(vWF). Plasma von Willebrand Factor (vWF) is a multimeric protein thatmediates adhesion of platelets to sites of vascular injury, andespecially the very large vWF multimers are haemostatically competent.The existence of plasma factors that control the size of vWF multimershas long been suspected. The von Willebrand Factor-cleaving protease(“vWF-cp”) is involved in the limitation of platelet thrombus growth byproteolytic cleavage of von Willebrand Factor multimers in man (Furlanet al., (1996) Blood 87: 4223-4234). Recently, the molecular structureof von Willebrand Factor-cleaving protease and the corresponding genehave been described (WO 02/42441; Zheng et al., (2001) J. Biol. Chem.276: 41059-41063) and have been identified as a new member of the ADAMTSfamily and designated ADAMTS13. vWFcp regulates vWF multimer size byproteolytic cleavage.

The large and ultra large vWF multimers play a central role in arterialthrombosis, whereby unusually large mutlimers of vWF have been seen intwo similar forms of thrombotic microangiopathy—thromboticthrombocytopenic purpura (TTP) and hemolytic-uremic syndrome (HUS)—bothresulting in formation of platelet aggregation leading to disseminatedocclusions in the microcirculation. Patients with TTP have a deficiencyof vWF-cp, whereas patients with HUS show normal activity of theprotease.

There are several types of TTP: An acute idiopathic or sporadic form, anintermittent form with an eventual relapse, and a chronic relapsingform. Chronic relapsing TTP is associated with acquired or congenitaldeficiency of vWF-cp. The rare hereditary form of TTP has been relatedto specific gene mutations in the ADAMTS-13 locus. Acute idiopathic TTPor acquired TTP is usually more severe than chronic relapsing TTP,wherein these patients have acquired antibodies against vWF-cp, whichinhibit the von Willebrand Factor-cleaving protease (Furlan et al.,(1998) Blood 91: 2839-2846; Furlan et al., (1998) N. Engl. J. Med. 339:1578-1584). Acquired TTP also occurs occasionally during pregnancy or inthe postpartum period. Intermittent relapsing TTP is also associatedwith the reappearance of vWF-cp inhibitor. For other forms of TTP, suchas ticlopidine-associated TTP, it has also been observed that thesepatients have acquired antibodies against vWF-cp (Moake, (2002) N. Eng.J. Med. 347:589-600). However, some patients with acquired TTP havingunusually large vWF multimers in plasma lack severe reduced levels ofvWF-cp.

In general, inhibitory antibodies against proteins cause seriousproblems, for example within the coagulation cascade, leading to bloodloss or thrombosis.

Congenital and acquired TTP are discriminated by the presence ofinhibitory antibodies against vWF-cp in the plasma of up to 80% ofpatients suffering from acquired TTP, and total absence of vWF-cp inplasma of patients with hereditary TTP. So far, inhibitory antibodies inplasma of patients are determined by static enzyme assays undernon-physiological conditions and confirm the diagnosis of acute,antibody-mediated TTP.

Different assays of vWF-cp for diagnosis of congenital and acquired TTPhave been described. vWF-cp activity and the presence of inhibitors ofvWF-cp are determined by incubation of purified vWF multimers withplasma samples of patients, followed by immunoblotting of degraded vWFsubstrate with anti-vWF antibodies and multimer analysis (Furlan et al.,(2002) Sem. Thromb. Haemost. 28:167-172). The method is very sensitivein the range of low protease activity; however, the accuracy is onlymoderate in the subnormal or normal range of protease activity. Acollagen-binding assay for determination vWF-cp activity and vWF-cpinhibitors as described by Gerritsen et al. [(1999) Thromb. Haemost.82:1386-1389] can be completed within 6 hours, but the method is lesssensitive in the very low range of protease activity as compared to theimmunoblotting of degraded vWF multimers (Furlan et al. 2002 supra). Theassays described in the prior art, however, are very cumbersome, timeconsuming and require the expertise of laboratories familiar with thetechnique. Moreover, the known prior art assays only allow for detectionof vWF-cp inhibitors that impair the catalytic function of vWF-cp.Inhibitory antibodies which may impair a vWF-cp function other than thecatalytic activity, e.g. endothelial cell binding, cannot be detected bythese assays.

Therefore a need exists for a test system that allows the detection anddetermination of anti-vWF-cp antibodies in a patient's plasma thatimpair vWF-cp function other than the enzyme's catalytic proteaseactivity.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a kit for determinationof an anti-vWF-cp antibody in a sample. The kit comprises vWF-cp and/orone or more vWF-cp fragment(s) immobilized on a solid phase withoutsubstantially impairing the biological property of the vWF-cp orvWF-cp-fragment(s). Additionally, the kit of the present invention mayalso contain any auxiliary agents known in the art for carrying outantigen/antibody assays (e.g., ELISA, EIA, RIA etc.), such as buffersalts, buffer disclosed solutions, blocking agents, detecting agents andthe like. The kits that are disclosed can be provided in a variety offormats, e.g., in the form of one or more containers or a microtiterplate.

Surprisingly, the inventors have found that vWF-cp or a vWF-cp fragmentimmobilized on a solid phase provides a simple, efficient, fast andreproducible assay system for determination of the presence of ananti-vWF-cp antibody in a sample. With the system of the presentinvention, vWF-cp inhibitors have been determined which were notdetected in a system of the prior art. The kit of the present inventionprovides an increased sensitivity in the current assay than prior artassays and can be used to detect vWF-antibodies amounts that may bebelow the detection limit of known systems. Assays performed with thekit of present invention allows one to discriminate between anti-vWF-cpantibodies having different specificities and based on impairment ofdifferent biological functions of vWF-cp. The assay to be performed withthe kit of the present invention further allows for a rapid diagnosis ofTTP and other disorders associated with vWF-cp inhibitors, as well asdifferentiation of various forms of thrombotic microangiopathy (TM).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows examples of plasmids that can be used for expression ofrecombinant vWF-cp, vWF-cp-fragment(s), or vWF-cp or vWF-cp-fragment(s)fused to a his-tag heterologous sequence.

FIGS. 2A and 2B show the determination of IgG (FIG. 2A) and IgM (FIG.2B) antibodies in plasma samples of a patient versus human normalplasma. The error bars indicate the two times added standard deviationof normal human plasma calculated from several plasma lots.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention relates to a kit for determination of ananti-vWF-cp antibody in a sample comprising vWF-cp and/or a vWF-cpfragment immobilized on a solid phase without substantially impairingthe biological property of the vWF-cp or the vWF-cp fragment. The vWF-cpor vWF-cp-fragment is used in the kit as diagnostic agent providing theantigenic determination site(s) capable of bindinganti-vWF-cp-antibodies present in a sample.

The term “determination” as used herein is meant to include detection,quantification and mapping of the vWF-cp antigen-binding region of ananti-vWF-cp-antibody in a sample. “Detection” means at least onepositive reaction indicating the formation of an antibody/vWF-cp—or anantibody/vWF-cp fragment—complex with a detection system, e.g., achromogenic assay. A sample known not to comprise any anti-vWF-antibody,e.g., normal human plasma is used as negative control. “Quantification”typically means that defined dilutions of a sample suspected to compriseanti-vWF-cp antibodies are contacted with the immobilized vWF-cp or avWF-cp fragment, and the intensity of the reaction obtained by thedetection system is compared to the intensity of the reaction obtainedwith defined dilutions of a sample comprising a known and defined amountof anti-vWF-antibodies, which is used as a standard. “Mapping” of thevWF-cp antigen binding site of an anti-vWF-cp antibody is performed bycontacting the sample suspected to comprise anti-vWF-cp antibodies withcomplete vWF-cp as well as with vWF-cp fragments derived from differentregions of the vWF-cp molecule. Thereby, the complete spectrum ofanti-vWF-cp antibodies possibly present in a sample can be captured andanti-vWF-cp antibodies having specific binding activity within aregion/domain of vWF-cp can be identified.

The term “sample” as used herein is meant to refer to a biological fluidsuch as blood, plasma or tissue of a patient. The sample may be inparticular obtained from human patients suspected of having a disorderassociated with occurrence of anti-vWF-cp antibodies

The term “solid phase” does not imply any specific limitations, andrelates, for example, to an unsoluble polymer material, which can be anorganic polymer, such as polyamide or a vinyl polymer (e.g.,poly(meth)acrylate, polystyrene and polyvinyl alcohol, or derivatesthereof), a natural polymer such as cellulose, dextrane, agarose, chitinand polyamino acids, or an inorganic polymer, such as glass ormetallohydroxide. The solid phase can be in the form of a microcarrier,particles, membranes, strips, paper, film, pearls or plates, such asmicrotiter plates. The vWF-cp or vWF-cp fragment(s) can be immobilizedon the solid phase directly by covalent coupling or via a carrier suchas a linker molecule or an antibody immobilized on the solid phase.

The term “biological property” as used herein is meant as functionallyactive epitopes or antigenic determinants of vWF-cp or the vWF-cpfragments, capable of binding at least one anti-vWF-cp antibody. Theimmobilization of vWF-cp or vWF-cp fragment on a solid phase isperformed in such a way that the immunologic properties, in particularthe structure of the functional epitopes and antigenic determinants ofvWF-cp or the vWF-cp fragments are preserved and efficiently presentedto be recognized by at least one anti-vWF-cp antibody present in thesample.

The vWF-cp or vWF-cp fragments can be produced in whole or in part byrecombinant techniques and can be prepared by expression in aprokaryotic or eukaryotic host system. Prokaryotic hosts can bebacterial cells such as E. coli or B. subtilis. Eukaryotic cells can beselected from the group consisting of yeast cells (e.g., Pichiastrains); insect cells (e.g., Sf9, Sf 21, High Five, S2); and mammaliancells, such as MRCS, CHO, COS, 3T3, HEK 293, BHK, SK-Hep, HepG2, CV-1,and Hela.

A wide variety of vectors can be used for the preparation of the vWF-cpor vWF-cp fragment(s) and can be selected from eukaryotic andprokaryotic expression vectors. Examples of vectors for prokaryoticexpression include plasmids such as pRSET, pET, pBAD, etc., wherein thepromoters used in prokaryotic expression vectors include lac, trc, trp,recA, araBAD, etc. Examples of vectors for eukaryotic expressioninclude: (i) for expression in yeast, vectors such as pAO, pPIC, pYES,pMET, using promoters such as AOX1, GAP, GAL1, AUG1, etc; (ii) forexpression in insect cells, vectors such as pMT, pAc5, pIB, pMIB, pBAC,etc., using promoters such as PH, p10, MT, Ac5, OpIE2, gp64, polh, etc.,and (iii) for expression in mammalian cells, vectors such as pSVL, pCMV,pRc/RSV, pcDNA3, pBPV, etc., and vectors derived form viral systems suchas vaccinia virus, adeno-associated viruses, herpes viruses,retroviruses, etc., using promoters such as CMV, SV40, EF-1a, UbC, RSV,ADV, BPV, and β-Actin.

The vWF-cp fragment(s) can be selected from the group consisting of SEQID NOs 1-6.

The vWF-cp fragments(s) can be peptides exhibiting amino acid sequencescontained in the vWF-cp and having preferably at least 6 amino acids,more preferably from about 6 to about 50 amino acids. One advantage ofusing said peptides as a diagnostic reagent in the present invention isthe selective determination of the specificity of the anti-vWF-cpantibody. The peptides can be produced by standard peptide synthesistechniques.

According to one embodiment of the invention, the vWF-cp or the vWF-cpfragment(s) are fused to a heterologous sequence. The heterologoussequence can be heterologous protein, polypeptide or peptide, inparticular a functional peptide. The heterologous sequence can be asequence having binding properties to a solid phase (e.g., the solidphase may have reactive site which allows covalent binding to theheterologous sequence, or has affinity to a carrier).

The heterologous protein, polypeptide or peptide can be selected fromthe group consisting of β-galactosidase, c-myc-product, glutathioneS-transferase, FLAG-tags and derivatives thereof. The heterologoussequence can also comprise a series of several equal or different aminoacids. Preferably, the heterologous sequence is a peptide that can forma covalent bond with the solid phase, or a polyhistidine that has highaffinity, particularly to specific anti-poly-histidine antibodies. Theheterologous sequence can be fused to vWF-cp or a vWF-cp fragment ateither its N- or C-terminus. The heterologous sequence is typicallyfused to the C-terminal end of vWF-cp. The vWF-cp or a vWF-cp fragmentis fused to the heterologous sequence such that the biological propertyof vWF-cp or a vWF-cp fragment is not negatively affected. A shortpeptide spacer may be inserted between the heterologous sequence andvWF-cp or a vWF-cp fragment, so as not to impede sterically thepresentation of the epitopes of vWF-cp or the vWF-cp fragment.

According to one embodiment, the vWF-cp or a vWF-cp fragment is fused toa functional affinity peptide, in particular a peptide having severalhistidine residues, in some instances 3 to 20 histidine residues, and inother instances 6 to 15 histidine residues. The use of an affinitypeptide in the form of poly-histidine (so called “His-tag”) C-terminallyfused to a protein for the purification of proteins has been describedin EP 0 282 042.

The immobilization on the solid phase can be effected (e.g., directly orby covalent binding) via reactive groups of the solid phase and theheterologous sequence, or via a carrier having affinity to theheterologous sequence.

In one preferred embodiment of the invention, the heterologous sequencehas high affinity to a carrier and the vWF-cp or vWF-cp fragment(s) areimmobilized on the solid phase via the binding of its heterologous partto the carrier. Accordingly, the heterologous sequence has specificbinding properties or high affinity to the carrier. According to oneembodiment of the invention, the carrier is an antibody having affinityto the heterologous part of the vWF-cp fusion protein.

In one embodiment of the invention, vWF-cp or a vWF-cp fragment is fusedto a poly histidine-tag as heterologous sequence and an anti-his-tagantibody is used as a carrier to immobilize vWF-cp or the vWF-cpfragment on a solid phase. Other heterologous affinity peptides andrespective anti-affinity-peptide antibodies known to the person skilledin the art can also be used to immobilize the vWF-cp or vWF-cp fragmentfusion protein.

The vWF-cp and/or vWF-cp fragment(s), or fusion proteins thereof, areimmobilized on the solid phase separately on different spots, e.g. indifferent wells of a microtiter plate, wherein typically one definedantigen such as vWF-cp or a specific vWF-fragment is contained in onespot. With this assay system, the complete spectrum of anti-vWF-cpantibodies can be captured and anti-vWF-cp antibodies having specificbinding activity within a region/domain of vWF-cp are identified. Thisis of major importance as by determination of anti-vWF-cp antibodyspecificity and determination of antigen binding site within the vWF-cpmolecule the whole range of antibodies can be identified, and a specifictreatment of patients having an anti-vWF-cp antibody associated disordercan be adapted, respectively. For example, anti-vWF-cp-antibodies can beselectively removed from the plasma of a patient identified to havespecific anti-vWF-cp antibodies by subjecting the patient's plasma toaffinity chromatography such as described herein which uses as anadsorbent specific vWF-cp fragments used in the assay and which haveaffinity to the antibody or antibodies. This allows for an improvedtreatment of patients having disorders associated with anti-vWF-cpantibodies compared to prior art methods.

According to one embodiment of the invention, the kit as described abovefurther comprises as diagnostic agent an anti-vWF-cp antibodyimmobilized on the solid phase. The anti-vWF-cp antibody can be amonoclonal antibody derived by conventional hybridoma techniques or canbe an antibody or antibody fragment obtained by recombinant technique,e.g., phage display or ribosome display. Such a set up in the kit of thepresent invention allows for differential diagnosis of thromboticmicroangiopathic disorders. In particular, by providing a kit comprisingimmobilized vWF-cp, vWF-cp fragment(s) and anti-vWF-cp antibody on asolid phase the presence/absence of anti-vWF antibodies as well as thepresence/absence of vWF-cp in a sample can be determined with one simpletest system.

The present invention is also related to a method for determination ofan anti-vWF-cp antibody in a sample, comprising the steps of providingvWF-cp and/or one or more vWF-cp fragment(s) immobilized on a solidphase without substantially impairing the biological property of thevWF-cp or vWF-cp fragment(s), contacting a biological sample of apatient suspected of having a disorder associated with the occurrence ofanti-vWF-cp antibody with the immobilized vWF-cp and/or one or morevWF-cp fragments, and detecting a complex of anti-vWF-cp antibody/vWF-cpand/or anti-vWF-cp antibody/vWF-cp fragment(s).

The complex of anti-vWF-cp antibody/vWF-cp or anti-vWF-cpantibody/vWF-cp fragment(s) can be detected by methods well known in theart, e.g. by detection with a labelled antibody. The detection methodcan be selected from the group consisting of an enzyme assay, achromogenic assay, a lumino assay, a fluorogenic assay, and aradioimmune assay. The reaction conditions to perform detection of theantibody/antigen-/complex formation depends upon the detection methodselected. It is within the knowledge of the person skilled in the art tochoose the optimal parameters, such as buffer system, temperature and pHfor the respective detection system to be used.

The invention also relates to a method for differential diagnosis ofthrombotic microangiopathic disorders with a kit as described above,wherein the kit comprises as diagnostic agent(s) either vWF-cp and/orone or more vWF-fragments, or vWF-cp and/or vWF-fragments andanti-vWF-cp antibodies, immobilized on a solid phase. The diagnosticagents are preferably each located on separate spots on the solid phase,e.g. in separate wells of a microtiter plate. This allows one todifferentiate between samples comprising either vWF-cp or anti-vWF-cpantibodies or both by one assay system and to differentiate betweenthrombotic microangiopathic disorders, e.g. different forms of TTP orHUS.

The kit and method of the present invention can be used for diagnosis ofa disorder associated with occurrence of anti-vWF-cp antibodies.

The kit and method of the present invention of the invention can also beused for diagnosis of different forms or disorders of thromboticmicroangiopathy. The thrombotic microangiopathic (TM) disorder can bethrombotic thrombocytic purpura (TTP), neonatal thrombocytopenia,Henoch-Schönlein purpura, preclampsia, or hemolytic—uremic syndrome(HUS), HELLP syndrome, ARDS, peripheral digit ischemic syndrome,nonocclusive mesenteric ischemia, acute pancreatitis, acute hepatitis,purpura rheumatica, medicament-associated formation of thrombocytopenia,post-operative TM, cancer-associated TM, disseminated intravascularcoagulation (DIC), systemic lupus erythematosus, liver cirrhosis,uremia, or acute inflammatory disorders.

The Examples provided herein clearly show that the presence of ananti-vWF-cp antibody in an acquired TTP patient, non-neutralizing in astandard vWF-cp activity assay but most likely impairing vWF-cp activityby mechanisms different from simply blocking substrate-cleavingactivity, can be determined using a kit and a method of the presentinvention. This allows the fast and sensitive diagnosis of TTP andurgent needed life-saving clinical intervention, i.e. plasma treatment.The kit and the method of the present invention can be used for thedifferential diagnosis of various forms of TTP.

With the kit and the method of the present invention, all IgG classes aswell as IgM antibodies can be detected, whereas prior art methods onlyallow detection of anti-vWF-cp antibodies of the IgG class.

The present invention will be further illustrated in the followingexamples, without any limitation thereto.

Example 1 Construction of a vWF-cp and vWF-cp Fragment/His(6×)-tag

For expression of vWF-cp protein the vWF-cp cDNA clone as described inWO 02/42442 is used.

To construct a vWF-cp his-tag fusion, two consecutive PCRs are carriedout to add the codons for 3× glycine, 6× histidines, stop and a XhoIrestriction site.

PCR1: the wild-type full length pcDNA3.1.(+)/vWF-cp (ADAMTS13) asdescribed in WO 02/42441 is used as template. With primers 7189 (5′ GTGATG GTG ATG GTG TCC ACC TCC GGT TCC TTC CTT TCC CTT CCA3′ (SEQ IDNO:19)) and 6526 (5′ CTG CCT CGC CCG GAA CCC CA 3′ (SEQ ID NO:20)) a 1.3kb fragment encompassing the C-terminal SgrAI/XhoI fragment frompcDNA3.1.(+)/vWF-cp is amplified. Using this fragment and primers 7190(5′ CCC TCT AGA CTC GAG TCA ATG GTG ATG GTG ATG GTG TCC ACC 3′ (SEQ IDNO:21)) and 6526, the second PCR is performed. The resulting product ispurified, digested with SgrAI and XhoI, and used to replace thecorresponding SgrAI/XhoI fragment in pcDNA3.1.(+)/vWF-cp wild-typeconstruct.

Using the full length vWF-cp cDNA clone disclosed in WO 02/42442 astemplate, vWF-cp fragment constructs containing different fragments ofthe gene of the mature protein are generated by PCR using the followingprimer combinations (see also Table 4 of Primers and respective vWF-cpdomain sequences).

E. coli Expression system: pBAD/Topo Thiofusion (Invitrogen)

Fusion: Thioredoxin (N-terminal), 6xHis-tail (C-terminal) DNA-fragmentprotein-fragment (bp) (aa) region in ADAMTS13  88-222 30(P)-74 (R)Propeptid  223-1317 75(A)-439(E) Cat./Disintegr./tsp1#1 1156-1317 386(R)-439 (E) Tsp1#1 1318-2055 440(K)-685(A) Cys-rich/spacer 2056-3393686(W)-1131(V) tsp1#2-8 3394-4281 1132(G)-1427(T) Cub1 + 2

The PCR fragments are cut with suitable restriction enzymes and clonedinto the vector such as pRSET (FIG. 1), and cleaved with the sameenzymes resulting in the desired plasmids.

For construction of vWF-cp fragment(s)-his tag fusions, the vWF-cpfragments are modified according to construction of vWF-cp/his-tag asdescribed above. The constructs are cloned with HIS-6 tag bysubstitution of the NdeI-XhoI fragment by the synthetic oligonucleotideso.pRET-FPdHIS(1)-6929 (SEQ ID NO:22) and o.pRSET-FPdHIS(2)-6930 (SEQ IDNO:23) (FIG. 1).

The vWF-cp, vWF-cp fragments or the respective his-tag fusions arerecombinantly expressed in E. coli JM 109, purified and used forimmobilization on a solid phase as described below.

HEK 293 Cell Clone Stably Expressing vWF-cp/C-His

HEK 293 (ATCC) cells are co-transfected with pcDNA3.1.(+)/vWF-cp/C-Hisand a selection plasmid harboring the hygromycine cassette using calciumphosphate precipitation. Initial clones and subsequent subclones areselected in culture medium supplemented with 100 μg/ml hygromycine and800 μg/ml G418 (neomycinphosphotransferase encoded on pcDNA).Recombinant expressed vWF-cp/his-tag is purified and used forimmobilization on a solid phase as described below.

Example 2 Coupling of vWF-cp and/or vWF-cp Fragment(s) on a Carrier

Recombinant vWFcp, vWF-cp fragment(s) are either coupled directly on asolid phase, or via monoclonal anti-vWF-cp antibodies as carriers.vWF-cp-His-tag or vWF-cp fragment-His-tag are immobilized via ananti-His tag antibody on the surface of an ELISA plate. After incubationwith a patient's plasma, anti-vWF-cp antibodies bound to vWF-cp orvWF-cp fragment are detected by a second antibody phosphatase conjugaterecognizing the constant human antibody region. The phosphatase reactedwith an appropriate substrate resulting in a chromogenic reaction and ayellow color. The intensity of the color is measured and the amount ofantibody in the sample is determined by comparison with a standard curvecomprising a known amount of anti-vWF antibody.

ELISA Setup:

A commercially available, BSA free, anti-His tag antibody(“carrier-antibody”; Qiagen, Germany) is diluted to a finalconcentration of 2 μg/mL in PBS pH 7.4. 100 μl per well is incubated forfour hours at room temperature in a 96 well-microtiter plate. Afterthree washing steps using PBST pH 7.4 (PBS buffer containing 0.1% (v/v)Tween 20), 250 μl of a blocking solution, containing PBS pH 7.4 and 2%(w/v) bovine serum albumin, are added and incubated at 4° C. over nightto block all free binding sites. The solution is replaced by 100 μl of arecombinant vWF-cp-His tag labelled preparation. vWF-cp concentration is1.5 μg/mL corresponding to 10 U/mL protease activity. vWF-cp samples arediluted to the final concentration in PBS 2% BSA. Due to the coatedanti-His antibody recombinant vWFcp/his-tag is captured and immobilizedvia the carrier antibody. After two hours at room temperature, tenwashing steps follow. The washing buffer contains PBS pH7.4 and 0.1%(v/v) Tween 20. Plasma samples of patients are diluted 1:20, 1:50,1:100, 1:200, 1:300, 1:400, 1:600, 1:800 and 1:1200 in PBS pH 7.4containing 2% BSA and 1000 of each dilution is incubated at roomtemperature for 3 hours on the recombinant vWF-cp-containing wells.Inhibitory antibodies are bound on the surface of the immobilized vWF-cpand unbound antibodies are washed away by ten washing steps using PBSTpH 7.4. Detection of human antibodies is performed with a mouseanti-human IgG Fc specific antibody or mouse anti-human IgM antibody,alkaline phosphatase conjugated. The antibody is diluted 1:60000 in PBS2% BSA to the final working solution and incubated for 2 hours at roomtemperature (100 μl/well), followed by ten washing steps with PBST pH7.4. Addition of an alkaline phosphatase substrate (pNPP) results in ayellow color, whereby the color intensity reflects the amount of boundantibody (antibody/vWF-cp). The color intensity is measured in an ELISAreader and the amount of antibody within the plasma sample is calculatedin reference to a standard curve of NP by serial dilution. As negativecontrol, dilutions of normal human plasma (NHP) are treated accordingly.The results are presented in FIGS. 2A and 2B. The results show thathuman anti-vWF-cp antibodies in a patients can be clearly detected in atleast a plasma dilution of 1:600.

Normal human plasma is used as control and the standard deviation (SD)calculated for several plasma lots. Antibody titres above that of normalhuman plasma+2 SD are evaluated as positive.

Analysis of TTP Patient Samples

Samples from patients with TTP and normal plasma samples are subjectedto ELISA comprising immobilized vWF-cp. The results are shown inTable 1. Patient 1 has an IgG titer of 1:600 and an IgM titer of 1:400.The IgG titer of patient 2 is much higher (1:1200) while the IgM titeris only 1:100. Patient 1 suffers from an acute TTP, while patient 2 isin remission after TTP. Patient 1 shows no inhibitory titer, whereaspatient 2 has an inhibitory titer of about 60 U/mL. Normal human plasmashows no reaction.

TABLE 1 Anti-vWF-cp antibody detection ELISA. IgG as well as IgM titersof two patients. 1:20 1:50 1:100 1:200 1:300 1:400 1:600 1:800 1:1200IgG#1 ++++ ++++ +++ +++ ++ ++ + − − IgM#1 +++ +++ +++ ++ ++ + − − − NP −− − − − − − − − IgG#2 ++++ ++++ ++++ +++ +++ +++ ++ ++ + IgM#2 ++ ++ + −− − − − − NP − − − − − − − − −

Samples of patients with TTP and normal plasma samples are subjected toELISA comprising immobilized vWF-cp fragments derived from differentregions of vWF-cp. The results are shown in Table 2. IgGs and IgMs ofpatient #1 (no inhibitory titer) show binding of antibodies on domainstrombospondin 2-8 and the Cub domains. IgGs and IgMs of patient #2 showbinding on the catalytic domain, which is consistent to the inhibitorytiter. Normal human plasma does not react with any domain. Patient'splasma is tested in duplicates and two different plasma dilutions (1:50and 1:100).

TABLE 2 Analysis of the binding on different ADAMTS-13 fragments ofpatient's antibodies Catalytic, Catalytic, Cys- Catalytic Catalyticdisintegrin, disintegrin, Cys-rich, rich, Tsp CUB CUB domain, domain,tsp1 tsp1 spacer, spacer, 2-8, Tsp 2-8, 1 + 2 1 + 2 1:50 1:100 1:501:100 1:50 1:100 1:50 1:100 1:50 1:100 IgG#1 − − − − − − ++ + + − IgM#1− − − − − − ++ + + − NP − − − − − − − − − − IgG#2 ++++ +++ ++ ++ − − − −− IgM#2 ++ ++ + − − − − − − NP − − − − − − − − −

Samples of patients with TTP and from normal plasma are subjected toELISA comprising immobilized anti-vWF-cp antibody. The results are shownin Table 3.

ADAMTS-13 levels of patients #1 and #2 can be clearly detected; normalhuman plasma shows the same levels. Patient #3 is being characterized tocarry a genetic defect on one allele causing a 50% reduced activity. A50% reduction on protein amount can also be seen in our assay system.Patient #4 is being characterized to completely lack ADAMTS-13 proteindue to a homozygous nonsense mutation. Consequently, no protein could bedetected.

TABLE 3 Detection of ADAMTS-13 levels in plasma using anti-vWF-cpantibodies for capturing. 1:20 1:50 1:100 1:200 1:300 1:400 1:600 1:8001:1200 ADAMTS-13 ++++ ++++ ++++ +++ +++ +++ ++ + − #1 ADAMTS-13 ++++++++ ++++ +++ +++ +++ ++ + − #2 ADAMTS-13 +++ +++ +++ ++ ++ + − − − #3ADAMTS-13 − − − − − − − − − #4 NP ++++ ++++ ++++ +++ +++ +++ ++ + −

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent or patent application were specifically andindividually indicated to be so incorporated by reference.

TABLE 4 PRIMER ADAMTS-13 (Baxter #) DNA sequence (5′→3′) DOMAINPRIMARY SEQUENCE 7442 (dp) CCCTCCCATTTCCAGCAGAGT Propeptid SEQ ID. 1:TGTCTT PSHFQQSCLQALEPQAVSSYLSPGAPLKGRPPSPGFQRQRQRQRR (SEQ ID NO: 7)7443 (rp) CCGCCTCTGCCTCTGCCTCTG (SEQ ID NO: 8) 7359 (rp)CTCGCAGGCCTGAGTGTTGCA Catalytic/ SEQ ID. 2: CATCTC disintegrin/AAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVH (SEQ ID NO: 9)Tsp-1/#1 LVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFKLEDPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGCTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSAGRARCVWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPLDQSSCSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRRWSSWGPRSPCSRSCGGGVVTPPPQCNNPRPAFGGRACVGADLQAEMCNTQACE 7360 (dp)GCTGCAGGCGGCATCCTACAC CTG (SEQ ID NO: 10) 7600 (dp)CGCTGGTCTAGCTGGGGTCCC Tsp-1//#1 SEQ ID. 3: (SEQ ID NO: 11)RWSSWGPRSPCSRSCGGGVVTPPPQCNNPRPAFGGRACVGADLQAEMCNTQAC E 7601 (rp)CTCGCAGGCCTGAGTGTTGCA (SEQ ID NO: 12) 7357 (rp) GGCCTGCCGTGGCTTAGGCTGCystein-rich/ SEQ ID. 4: GAAGTA spacerKTQLEFMSQQCARTDCQPLRSSPGGASFYHWGAAVPHSQGDALCRHMCRAIGE (SEQ ID NO: 13)SFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDCRMDSQQVWDRCQVCGGDNSTCSPRKGSFTAGRAREYCTFLTCTPNLTSCYIANHRPLFTHLAVRIGGRYVVAGKMSISPNTTYPSILLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRYGEEYGNLTRPDITFTYFQPKPRQA 7358 (dp) AAGACCCAGCTGGAGTTCATGTCGCAA (SEQ ID NO: 14) 7441 (dp) TGGGTGTGGGCCGCTGTGCGT Tsp-1/#2-8SEQ ID. 5: (SEQ ID NO: 15)WVWAAVRGPCSVSSGAGLRWVNQSCLDQARKELVETVQCQGSQQPPAWPEACVLEPCPPYWAVGDFGPCSASCGGGLRERPVRCVEAQGSLLKTLPPARCRAGAQQPAVALETCNPQPCPARWEVSEPSSCTSAGGAGLALENETCVPGADGLEAPVTEGPGSVDEKLPAPEPCVGMSCPPGWGHLDATSAGEKAPSPWGSIRTGAQAAHVWTPVAGSCSVSCGRGLMELRFLCMDSALRVPVQEELCGLASKPGSRREVCQAVPCPARWQYKLAACSVSCGRGVVRRILYCARAHGEDDGEEILLDTQCQGLPRPEPQEACSLEPCPPRWKVMSLGPCSASCGLGTARRSVACVQLDQGQDVEVDEACAALVRPEASVPCLIADCTYRWHVGWMECSVSCGDGIQRRRDTCLGPQAQAPVPADFCQHLPKPVTVRGCWAGPCV 7444 (rp) CACACAGGGCCCAGCCCAGCA (SEQ ID NO: 16)7439 (dp) GGACAGGGTACGCCCAGCCTG Cub 1 + 2 SEQ ID. 6: (SEQ ID NO: 17)GQGTPSLVPHEEAAAPGRTTATPAGASLEWSQARGLLFSPARQPRRLLPGPQENSVQSSACGRQHLEPTGTIDMRGPCQADCAVAIGRPLGEVVTLRVLESSLNCSAGDMLLLWGRLTRKMCRKLLDMTFSSKTNTLVVRQRCGRPGGGVLLRYGSQLAPETFYRECDMQLFGPWGEIVSPSLSPATSNAGGCRLFINVAPHARIAIHALATNMGAGTEGANASYILIRDTHSLRTTAFHGQQVLYWESESSQAEMEFSEGFLKAQALRGQYWTLQSWVPEMQDPQSWKGKEGT 7440 (rp) GGTTCCTTCCTTTCCCTTCCA GGACTG(SEQ ID NO: 18)

1. A kit for detecting an anti-von Willebrand Factor-cleaving protease(“anti-vWF-cp”) anti-vWF-cp antibody in a sample, where the antibodybinds to a vWF-cp domain selected from the group consisting of acatalytic domain, a thrombospondin 2-8 domain, and a Cub domain, the kitcomprising a solid phase comprising the vWF-cp domain, which isimmobilized and fused to a heterologous sequence that is separated fromthe vWF-cp domain by a spacer; wherein the biological property of saidvWF-cp domain is not substantially impaired and the presentation of theepitopes of the vWF-cp domain is not sterically impeded.
 2. The kitaccording to claim 1, wherein the solid phase is selected from the groupconsisting of plates, membranes, paper, film, strips, and pearls.
 3. Thekit according to claim 1, wherein said vWF-cp domain is immobilized onthe solid phase via a carrier.
 4. The kit according to claim 3, whereinsaid carrier is an antibody.
 5. The kit according to claim 4, whereinsaid antibody is directed to the heterologous sequence fused to saidvWF-cp fragment.
 6. The kit according to claim 1, further comprising anadditional vWF-cp domain selected from the group consisting of acatalytic domain, a thrombospondin 2-8 domain, and a Cub domain, whereinthe second domain is separately arranged at a different spot on thesolid phase.
 7. The kit according to claim 1, further comprising ananti-vWF-cp antibody immobilized on said solid phase.
 8. The kitaccording to claim 7, wherein said vWF-cp domain and anti-vWF-cpantibody are each separately arranged in different spots on the solidphase.